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The ?? and ?? Classes Carbonic Anhydrases from Helicobacter pylori as Novel Drug Targets
Abstract
Helicobacter pylori (H. pylori) successfully resides in the human stomach in highly acidic conditions, causing a variety of gastroduodenal lesions, including gastric ulcer, gastric cancer and MALT lymphoma. For acid acclimation of H. pylori, two types of enzymes, urease and carbonic anhydrase (CA), play a central role. They cooperatively function to maintain neutral pH in the bacterial cytoplasm and periplasm. The genome project of H. pylori identified two different classes of CA with different subcellular localization: a periplasmic class CA (hp alpha CA) and a cytoplasmic beta-class CA (hp beta CA). These two CAs are catalytically efficient with almost identical activity to that of the human isoform CA I for the CO2 hydration reaction, and highly inhibited by many sulfonamides/sulfamates, including acetazolamide, ethoxzolamide, topiramate and sulpiride, all clinically used drugs. Furthermore, certain CA inhibitors, such as acetazolamide and methazolamide, were shown to inhibit the bacterial growth in vitro. Since the efficacy of eradication therapies currently employed has been decreasing due to drug resistance and side effects of the commonly used drugs, the dual inhibition of alpha- and/or beta-CAs of H. pylori could be applied as an alternative therapy in patients with H. pylori infection or for the prevention of gastroduodenal diseases provoked by this widespread pathogen.
... [7b] In the last few years, numerous reports on pathogenic bacterial CAs in vitro inhibition profiles were produced. [2,[9][10][11][12][13][14] Among the strains explored, Helicobacter pylori, Mycobacterium tuberculosis, Vibrio cholerae, Streptococcus mutans, Burkholderia pseudomallei, Legionella pneumophila, Clostridium perfringens, and Porphyromonas gingivalis were the most important. [2,[9][10][11][12] All the investigations carried out on these microorganisms were aimed to identify new CA inhibitors (CAIs) endowed with potential antibacterial activity. ...
... [2,[9][10][11][12][13][14] Among the strains explored, Helicobacter pylori, Mycobacterium tuberculosis, Vibrio cholerae, Streptococcus mutans, Burkholderia pseudomallei, Legionella pneumophila, Clostridium perfringens, and Porphyromonas gingivalis were the most important. [2,[9][10][11][12] All the investigations carried out on these microorganisms were aimed to identify new CA inhibitors (CAIs) endowed with potential antibacterial activity. [9][10][11][12][13][14] Although this study has the same scientific objectives, we propose a series of compounds obtained by means of a modular design strategy though to grant: i) easy access to variegate chemical diversities potentially useful for the identification of valuable CAIs as well as for structure-activity relationship (SAR) refinements; ii) low cost and high efficient scale-up syntheses of selected products. ...
... [2,[9][10][11][12] All the investigations carried out on these microorganisms were aimed to identify new CA inhibitors (CAIs) endowed with potential antibacterial activity. [9][10][11][12][13][14] Although this study has the same scientific objectives, we propose a series of compounds obtained by means of a modular design strategy though to grant: i) easy access to variegate chemical diversities potentially useful for the identification of valuable CAIs as well as for structure-activity relationship (SAR) refinements; ii) low cost and high efficient scale-up syntheses of selected products. All compounds obtained were investigated in vitro for their inhibition against the recombinant CAs from V. cholerae (i. ...
A series of benzylaminoethylureido‐tailed benzenesulfonamides was analyzed for their inhibition potential against bacterial carbonic anhydrases (CAs) such as VhCA α, β, and γ from Vibrio cholerae, and BpsCA β and γ‐CAs from Burkholderia pseudomallei. Growing drug resistance against antibiotics demands alternative targets and mechanisms of action. As CA is essential for the survival of bacteria, such enzymes have the potential for developing new antibiotics. Most of the compounds presented excellent inhibition potential against VhCA γ compared to α and β, with Ki values in the range of 82.5–191.4 nM. Several sulfonamides exhibited excellent inhibition against BpsCA β with Ki values in the range of 394–742.8 nM. Recently it has been demonstrated that sufonamide CA inhibitors are effective against vancomycin‐resistant enterococci. These data show that CA inhibition of pathogenic bacteria may lead to a new class of antibiotics.
... After the 1990s, many new β-CAs were discovered in the genomes of various organisms [11]. Based on current knowledge, these enzymes are found in photosynthetic organisms, eubacteria, yeasts, and Archaea [11][12][13]. Later on, it was discovered that they are also present in the genomes of insects, nematodes, and protozoans, but not in mammals [14]. Therefore, β-CAs are considered promising target enzymes for antiparasitic drugs [15][16][17]. ...
... However, β-CAs were shown to have important roles (for instance, in providing bicarbonate/CO 2 for the photosynthetic enzyme Rubisco in the chloroplasts of many plants/algae [11,17,18]). Helicobacter pylori contains only one αand one β-CA, whose inhibition with sulfonamides impairs the growth of the pathogen in vitro and in vivo [13,15]. The physiological relevance of β-CAs in many organisms, including protozoans belonging to the Amoebozoas, is yet to be discovered. ...
... It can be observed that, similar to hCA I, EhiCA was inhibited in the high nanomolar range by this compound, with an inhibition constant K I s of 509 nM (Table 1). The dimer and the trimer of EhiCA are also seen (arrows), as reported for other β-CAs cloned and purified earlier [11][12][13][14][15]. ...
We report the cloning and catalytic activity of a β-carbonic anhydrase (CA, EC 4.2.1.1), isolated from the pathogenic protozoan Entamoeba histolytica, EhiCA. This enzyme has a high catalytic activity for the physiologic CO2 hydration reaction, with a kcat of 6.7 × 105 s−1 and a kcat/Km of 8.9 × 107 M−1 × s−1. An anion inhibition study of EhiCA with inorganic/organic anions and small molecules revealed that fluoride, chloride, cyanide, azide, pyrodiphosphate, perchlorate, tetrafluoroborate and sulfamic acid did not inhibit the enzyme activity, whereas pseudohalides (cyanate and thiocyanate), bicarbonate, nitrate, nitrite, diethyldithiocarbamate, and many complex inorganic anions showed inhibition in the millimolar range (KIs of 0.51–8.4 mM). The best EhiCA inhibitors were fluorosulfonate, sulfamide, phenylboronic acid and phenylarsonic acid (KIs in the range of 28–86 μM). Since β-CAs are not present in vertebrates, the present study may be useful for detecting lead compounds for the design of effective enzyme inhibitors, with potential to develop anti-infectives with alternative mechanisms of action.
... Helicobacter pylori is able to survive the acidic pH in the stomach thanks to, at least, two enzymes playing a fundamental role in acid acclimation. Urease and carbonic anhydrase maintain neutral pH in the bacterial cytoplasm and periplasm by converting urea and carbon dioxide into ammonia and bicarbonate [54,55,67]. Urease, which is absent in humans, is a critical enzyme for Hp colonization of the host stomach. ...
... On the other hand, two different types of carbonic anhydrase, αand β-, have been identified in Hp periplasm and cytoplasm, respectively. Both metalloenzymes have been described to be essential for acid acclimation, biosynthetic reactions, bacterial survival, and colonization of the stomach and duodenum [67,68,[70][71][72]. They are targeted by sulphonamide antimicrobial agents and phenol-derivatives [68,69,71]. ...
Flavodoxins are small soluble electron transfer proteins widely present in bacteria and absent in vertebrates. Flavodoxins participate in different metabolic pathways and, in some bacteria, they have been shown to be essential proteins representing promising therapeutic targets to fight bacterial infections. Using purified flavodoxin and chemical libraries, leads can be identified that block flavodoxin function and act as bactericidal molecules, as it has been demonstrated for Helicobacter pylori (Hp), the most prevalent human gastric pathogen. Increasing antimicrobial resistance by this bacterium has led current therapies to lose effectiveness, so alternative treatments are urgently required. Here, we summarize, with a focus on flavodoxin, opportunities for pharmacological intervention offered by the potential protein targets described for this bacterium and provide information on other gastrointestinal pathogens and also on bacteria from the gut microbiota that contain flavodoxin. The process of discovery and development of novel antimicrobials specific for Hp flavodoxin that is being carried out in our group is explained, as it can be extrapolated to the discovery of inhibitors specific for other gastric pathogens. The high specificity for Hp of the antimicrobials developed may be of help to reduce damage to the gut microbiota and to slow down the development of resistant Hp mutants.
... In the previous work [1,2] we also investigated the inhibition profile of the new enzyme (nominated EhiCA) with the main classes of CA inhibitors (CAIs) [7][8][9][10], the sulfonamides and the inorganic anions [11][12][13][14]. Our main scope was to identify agents that by interference with the activity of this enzyme, might lead to anti -infectives with a novel mechanism of action, considering the fact that many CAs are essential in the life cycle of microorganisms belonging to the bacteria, fungal or protozoan domains [15][16][17]. As β-CAs are not present in mammals [18,19], effective EhiCA inhibitors may represent an alternative therapeutic option for this protozoan infection. ...
... Indeed, various pathogenic organisms belonging to the bacteria, fungal or protozoan domains encode for CAs, which have been investigated in some detail ultimately, in the search of antiinfectives with a diverse mechanism of action [7][8][9][10][14][15][16][17][18][19][20][21][22][23]. CAs catalyze the reaction between CO2 and water, with formation of bicarbonate (HCO3 -) and protons (H + ), and are highly effective catalysts, among the most efficient known so far in nature [7][8][9][10]. ...
The β-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic protozoan Entamoeba histolytica, EhiCA, was investigated for its activation with a panel of natural and non-natural amino acids and amines. EhiCA was potently activated by D-His, D-Phe, D-DOPA, L- and D-Trp, L- and D-Tyr, 4-amino-L-Tyr, histamine and serotonin, with KAs ranging between 1.07 and 10.1 M. The best activator was D-Tyr (KA of 1.07 µM). L-Phe, L-DOPA, L-adrenaline, L-Asn, L-Asp, L-Glu and L-Gln showed medium potency activation, with KAs of 16.5–25.6 µM. Some heterocyclic- alkyl amines, such as 2-pyridyl-methyl/ethyl-amine and 4-(2-aminoethyl)-morpholine, were devoid of EhiCA activating properties with KAs > 100 µM. As CA activators have poorly been investigated for their interaction with protozoan CAs, our study may be relevant for an improved understanding of the role of this enzyme in the life cycle of E. histolytica.
... CAs belonging to, at least, three classes were found in the genomes of many pathogenic bacteria including Helicobacter pylori (Nishimori et al., 2008), Vibrio cholerae (Del Prete et al., 2012), Mycobacterium tuberculosis (Nishimori et al., 2010), and most of them are necessary for the growth of pathogens which causes diseases (De Luca et al., 2016). CA inhibitors (CAIs) targeting such pathogenic enzymes have been reported in recent years as an alternative to antibacterials to which pathogens are resistant (Supuran, 2011). ...
The synthesis and biological assessment of novel multi-functionalized pyrrolidine-containing benzenesulfonamides were reported along with their antimicrobial, antifungal, CAs inhibition, and AChE inhibition as well as DNA-binding effects. The chemical structure of the compounds was elucidated by using FTIR, NMR, and HRMS. Compound 3b, which had Ki values of 17.61 ± 3.58 nM (hCA I) and 5.14 ± 0.61 nM (hCA II), was found the be the most potent CAs inhibitor. Compounds 6a and 6b showed remarkable AChE inhibition effects with Ki values 22.34 ± 4.53 nM and 27.21 ± 3.96 nM in comparison to tacrine. Compounds 6a-6c had moderate antituberculosis effect on M. tuberculosis with a MIC value of 15.62 μg/ml. Compounds had weaker antifungal and antibacterial activity in the range of MIC 500-62.5 μg/ml against standard bacterial and fungal strains. Besides these above, molecular docking studies were performed to examine and evaluate the interaction of the remarkable compounds (3b, 6a and 6b) against the current enzymes (CAs and AChE). Novel compounds gained interest in terms of enzyme inhibitory potencies. Therefore, the most potent enzyme inhibitors may be considered lead compounds to be modified for further research.Communicated by Ramaswamy H. Sarma.
... The above-described data indicate that canB was essential in A. baumannii; therefore, chemical inhibition of CanB may lead to cell death. To test this, we evaluated a known carbonic anhydrase inhibitor, ethoxzolamide (EZA), for effects on cell growth (Table 5) (70)(71)(72)(73). We initially tested EZA against wild-type ATCC 17978, but no growth inhibition was observed. ...
The ADC (AmpC) β-lactamase is universally present in the Acinetobacter baumannii chromosome, suggesting it may have a yet-to-be-identified cellular function. Using peptidoglycan composition analysis, we show that overexpressing the ADC-7 β-lactamase in A. baumannii drives changes consistent with altered l,d-transpeptidase activity. Based on this, we tested whether cells overexpressing ADC-7 would exhibit new vulnerabilities. As proof of principle, a screen of transposon insertions revealed that an insertion in the distal 3' end of canB, encoding carbonic anhydrase, resulted in a significant loss of viability when the adc-7 gene was overexpressed. A canB deletion mutant exhibited a more pronounced loss of viability than the transposon insertion, and this became amplified when cells overexpressed ADC-7. Interestingly, overexpression of the OXA-23 or TEM-1 β-lactamases also led to a pronounced loss of viability in cells with reduced carbonic anhydrase activity. In addition, we demonstrate that reduced CanB activity led to increased sensitivity to peptidoglycan synthesis inhibitors and to the carbonic anhydrase inhibitor ethoxzolamide. Furthermore, this strain exhibited a synergistic interaction with the peptidoglycan inhibitor fosfomycin and ethoxzolamide. Our results highlight the impact of ADC-7 overexpression on cell physiology and reveal that the essential carbonic anhydrase CanB may represent a novel target for antimicrobial agents that would exhibit increased potency against β-lactamase-overexpressing A. baumannii. IMPORTANCE Acinetobacter baumannii has become resistant to all classes of antibiotics, with β-lactam resistance responsible for the majority of treatment failures. New classes of antimicrobials are needed to treat this high-priority pathogen. This study had uncovered a new genetic vulnerability in β-lactamase-expressing A. baumannii, where reduced carbonic anhydrase activity becomes lethal. Inhibitors of carbonic anhydrase could represent a new method for treating A. baumannii infections.
... 10 The α-CA from Helicobacter pylori is essential for survival in the acidic environment in the stomach, and CA inhibitors (CAIs) show activity against H. pylori. 11,12 Enterococcus faecium and Enterococcus faecalis express both αand γ-CA isoforms that appear to be essential and are susceptible to CAIs. 13 Mycobacterium tuberculosis, 14 Vibrio cholera, 15 Burkholde-Burkholderia species, 16 Brucella suis, 17 and many other bacteria also express CAs, with further research necessary to determine the essentiality in each. 18 N. gonorrhoeae is susceptible to human CAIs, including acetazolamide (AZM), 19 and expresses three sub-classes of carbonic anhydrases, with the α-CA (α-NgCA) proven to be essential for bacterial viability. ...
Drug-resistant Neisseria gonorrhoeae is a critical threat to public health, and bacterial carbonic anhydrases expressed by N. gonorrhoeae are potential new therapeutic targets to combat this pathogen. To further expand upon our recent reports of bacterial carbonic anhydrase inhibitors for the treatment of N. gonorrhoeae, our team has solved ligand-bound crystal structures of the FDA-approved carbonic anhydrase inhibitor acetazolamide, along with three analogs, in complex with the essential α-carbonic anhydrase isoform from N. gonorrhoeae. The structural data for the analogs presented bound to N. gonorrhoeae α-carbonic anhydrase supports the observed structure-activity relationship for in vitro inhibition with this scaffold against the enzyme. Moreover, the ligand-bound structures indicate differences in binding poses compared to those traditionally observed with the close human ortholog carbonic anhydrase II. These results present key differences in inhibitor binding between N. gonorrhoeae α-carbonic anhydrase and the human carbonic anhydrase II isoform.
... The Bacteria domain has three class (α, β, and γ) of CA enzymes, while the Archaea domain has two class (β and γ) of CA enzymes Nishimori et al., 2008). In the active side of this enzyme (α, β and γ) a zinc ion is found and coordinated by one histidine (His) and two cysteine (Cys) residues (β class) or by three His residues and a water molecule/hydroxide ion (α and γ) and the fourth ligand is a water molecule/hydroxide ion (Fig. 1). ...
In this study, the characterization and inhibition characteristic of α-class carbonic anhydrase from Chromohalobacter (ChCA) was documented for the first time. The carbonic anhydrase enzyme had 47.77% yield and 54.45-fold purity. The specific activity of the enzyme was determined as 318.52 U/mg proteins. Alternative substrate (4-nitrophenyl trifluoroacetate, 4-nitrophenyl phosphate, 4-nitrophenyl sulphate and 4-nitrophenyl acetate) were tested for the enzyme. KM and Vmax values for 4-nitrophenyl acetate were 4.57 mM and 4.29 EU/mL and for 4-nitrophenyl trifluoroacetate were 2.39 mM and 2.41 EU/mL. The anions, Cl⁻, NO2⁻, NO3⁻, Br⁻, ClO3⁻, ClO4⁻, I⁻, CO3²⁻ and SO4²⁻, inhibited the ChCA hydratase activity. Among nine anions, the strongest inhibitor activities were obtained with micro molar concentrations of NO2⁻, NO3⁻, Br⁻, I⁻, CO3²⁻ (KI values of 160–255 μM). Other four anions tested (Cl⁻, ClO3⁻, ClO4⁻ and SO4²⁻) showed moderate inhibitory activities (KI values of 680–813.5 μM). The results obtained demonstrate that the anions we tested inhibit the Chromohalobacter CA (ChCA) enzyme as in other α-CAs in mammals; however, the susceptibility of ChCA resulted from anions differed significantly from that of other organism CAs.
... These different classes of CAs have different structural folds and share low sequence homology [7,8]. α-CAs are the most studied CAs, in which Zn 2+ ions are present as centres in their active sites, coordinated to three histidines residues [9][10][11]. In humans, CAs are vital for physiological processes such as regulation of blood pH, regulation of pressure of retinal fluids, and nourishment during bone growth [12]. ...
Molecular basis of protein stability at different temperatures is a fundamental problem in protein science that is substantially far from being accurately and quantitatively solved as it requires an explicit knowledge of the temperature dependence of folding free energy of amino acid residues. In the present study, we attempted to gain insights into the thermodynamic stability of SazCA and its implications on protein folding/unfolding. We report molecular dynamics simulations of water solvated SazCA in a temperature range of 293-393 K to study the relationship between the thermostability and flexibility. Our structural analysis shows that the protein maintains the highest structural stability at 353 K and the protein conformations are highly flexible at temperatures above 353 K. Larger exposure of hydrophobic surface residues to the solvent medium for conformations beyond 353 K were identified from H-bond analysis. Higher number of secondary structure contents exhibited by SazCA at 353 K corroborated the conformations at 353 K to exhibit the highest thermal stability. The analysis of thermodynamics of protein stability revealed that the conformations that denature at higher melting temperatures tend to have greater maximum thermal stability. Our analysis shows that 353 K conformations have the highest melting temperature, which was found to be close to the experimental optimum temperature. The enhanced protein stability at 353 K due the least value of heat capacity at unfolding suggested an increase in folding. Comparative Gibbs free energy analysis and funnel shaped energy landscape confirmed a transition in folding/unfolding pathway of SazCA at 353 K.
... The bacterial extracellular vesicles (EVs) are generated in a budding manner similar to that of the yeasts (Kim et al., 2015). Gram-negative bacteria, differently from the Gram-positive bacteria, produce extracellular vesicles by pinching off the outer membrane and, for this reason, are defined with the a Human/bacterial recombinant isozymes and stopped-flow CO 2 hydrase assay method, as reported in Nishimori et al. (2008). b Recombinant hpCA and stopped-flow CO 2 hydrase assay method, as reported in this work, mean ± SE (from three different assays) and in Nishimori et al. (2007). ...
Our understanding of the function of bacterial carbonic anhydrases (CAs, EC 4.2.1.1) has increased significantly in the last years. CAs are metalloenzymes able to modulate CO 2 , HCO 3 – and H ⁺ concentration through their crucial role in catalysis of reversible CO 2 hydration (CO 2 + H 2 O ⇄ HCO 3 – + H ⁺ ). In all living organisms, CA activity is linked to physiological processes, such as those related to the transport and supply of CO 2 or HCO 3 – , pH homeostasis, secretion of electrolytes, biosynthetic processes and photosynthesis. These important processes cannot be ensured by the very low rate of the non-catalyzed reaction of CO 2 hydration. It has been recently shown that CAs are important biomolecules for many bacteria involved in human infections, such as Vibrio cholerae , Brucella suis , Salmonella enterica , Pseudomonas aeruginosa , and Helicobacter pylori . In these species, CA activity promotes microorganism growth and adaptation in the host, or modulates bacterial toxin production and virulence. In this review, recent literature in this research field and some of the above-mentioned issues are discussed, namely: ( i ) the implication of CAs from bacterial pathogens in determining the microorganism growth and virulence; ( ii ) the druggability of these enzymes using classical CA inhibitors (CAIs) of the sulfonamide-type as examples; ( iii ) the role played by Helicobacter pylori CAs in the acid tolerance/adaptation of the microbe within the human abdomen; ( iv ) the role of CAs played in the outer membrane vesicles spawned by H. pylori in its planktonic and biofilm phenotypes; ( v ) the possibility of using H. pylori CAIs in combination with probiotic strains as a novel anti-ulcer treatment approach. The latter approach may represent an innovative and successful strategy to fight gastric infections in the era of increasing resistance of pathogenic bacteria to classical antibiotics.
... These pathogenic features are in part due to its bacillary and curved s-shape (spiral) form plus several flagella that confer with its high mobility. Also, H. pylori possesses oxidase, catalase, urease, and carbonic anhydrase (Mobley et al., 1991;Takeuchi et al., 2008). These enzymes help to neutralize the acidic environment of the stomach, allowing H. pylori to survive for decades in the gastric epithelium (Wroblewski et al., 2010;Skoog et al., 2012). ...
The full UHPLC-MS metabolome fingerprinting and anti-Helicobacter pylori effect of Gunnera tinctoria (Molina) Mirb. (Nalca) total extract (GTE) and fractions prepared from its edible fresh petioles were evaluated. The activity of G. tinctoria against H. pylori strains ATCC 45504 and J99 was assessed in vitro by means of agar diffusion assay, Minimum Inhibition Concentration (MIC), and Minimum Bactericidal Concentration (MBC), while killing curve and transmission electronic microscopy (TEM) were conducted in order to determine the effect of the plant extract on bacterial growth and ultrastructure. Additionally, the inhibitory effect upon urease was evaluated using both the Jack Bean and H. pylori enzymes. To determine which molecules could be responsible for the antibacterial effects, tentative identification was done by ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-Q-Orbitrap®-HR-MS). Furthermore, the total G. tinctoria extract was fractionated using centrifugal partition chromatography (CPC), giving four active fractions (1–4). It was determined that the crude extract and centrifugal partition chromatography fractions of G. tinctoria have a bactericidal effect being the lowest MIC and MBC = 32 μg/ml. In the killing curves, fraction one acts faster than control amoxicillin. In the urease assay, F3 exhibited the lowest IC50 value of 13.5 μg/ml. Transmission electronic microscopy showed that crude G. tinctoria extract promotes disruption and separation of the cellular wall and outer membrane detachment on H. pylori causing bacterial cell death.
... Although there is no sequence homology between the families of CAs, they all contain zinc ions (Zn 2+ ) as metal centers in their active sites. [8][9][10][11][12][13][14] Due to the association of CO 2 with the reaction they catalyze, they have been reported as potential candidates in carbon capture, storage, and sequestration (CCUS). 15 Unfortunately, the use of CAs in CCUS is generally limited by their poor stability under reactor operation conditions. ...
The present study examined the structure and dynamics of the most active and ther-mostable carbonic anhydrase, SazCA, probed using molecular dynamics simulations. The molecular system was described by widely used biological force-fields (AMBER, CHARMM22, CHARMM36, and OPLS-AA) in conjunction with TIP3P water model. The comparison of molecular dynamics simulation results suggested AMBER to be a suitable choice to describe the structure and dynamics of SazCA. In addition to this, we also addressed the effect of temperature on the stability of SazCA. We performed molecular dynamics simulations at 313, 333, 353, 373, and 393 K to study the relationship between thermostability and flexibility in SazCA. The amino acid residues VAL98, ASN99, GLY100, LYS101, GLU145, and HIS207 were identified as the most flexible residues from root-mean-square fluctuations. The salt bridge analysis showed that ion-pairs ASP113-LYS81, ASP115-LYS81, ASP115-LYS114, GLU144-LYS143, and GLU144-LYS206, were responsible for the compromised thermal stability of SazCA. K E Y W O R D S carbon capture, carbonic anhydrase, molecular simulations, stability, thermostable protein
... In addition, it was shown to be effective against clinical isolates resistant to conventional antibiotics, suggesting that EZA kills H. pylori via mechanisms different from those of metronidazole, clarithromycin, and amoxicillin. Developed as an inhibitor of hCAs, EZA is also known to inhibit activity of H. pylori α-and β-carbonic anhydrases (HpαCA and HpβCA) [12,13]. The crystal structures of HpαCA bound to EZA and related sulfonamides demonstrated that these compounds act as competitive inhibitors mimicking the transition state of the reaction, and that their structure correlates well with their in vitro inhibitory properties [14,15]. ...
Abstract With the rise of bacterial resistance to conventional antibiotics, re-purposing of Food and Drug Administration (FDA) approved drugs currently used to treat non-bacteria related diseases as new leads for antibacterial drug discovery has become an attractive alternative. Ethoxzolamide (EZA), an FDA-approved diuretic acting as a human carbonic anhydrase inhibitor, is known to kill the gastric pathogenic bacterium Helicobacter pylori in vitro via an, as yet, unknown mechanism. To date, EZA activity and resistance have been investigated for only one H. pylori strain, P12. We have now performed a susceptibility and resistance study with H. pylori strains SS1 and 26695. Mutants resistant to EZA were isolated, characterized and their genomes sequenced. Resistance-conferring mutations were confirmed by backcrossing the mutations into the parent strain. As with P12, resistance to EZA in strains SS1 and 26695 does not develop easily, since the rate of spontaneous resistance acquisition was less than 10−8. Acquisition of resistance was associated with mutations in 3 genes in strain SS1, and in 6 different genes in strain 26695, indicating that EZA targets multiple systems. All resistant isolates had mutations affecting cell wall synthesis and control of gene expression. EZA’s potential for treating duodenal ulcers has already been demonstrated. Our findings suggest that EZA may be developed into a novel anti-H. pylori drug.
... The increasing resistance to first-line antibiotic drugs, especially metronidazole and clarithromycin [55,56], has had a dramatic impact on the eradication rates, which have fallen to 70% in the last few years [1]. In order to overcome the antimicrobial resistance strategies evolved by this pathogen, novel molecular targets have emerged as candidates for therapeutic interventions [23,24,[57][58][59][60][61]. In a previous work, we validated a new effective anti-H. ...
Antibiotic resistance is a major cause of the increasing failures in the current eradication therapies against Helicobacter pylori. In this scenario, repurposing drugs could be a valuable strategy to fast-track novel antimicrobial agents. In the present study, we analyzed the inhibitory capability of 1,4-dihydropyridine (DHP) antihypertensive drugs on the essential function of the H. pylori response regulator HsrA and investigated both the in vitro antimicrobial activities and the in vivo efficacy of DHP treatments against H. pylori. Six different commercially available and highly prescribed DHP drugs—namely, Nifedipine, Nicardipine, Nisoldipine, Nimodipine, Nitrendipine, and Lercanidipine—noticeably inhibited the DNA binding activity of HsrA and exhibited potent bactericidal activities against both metronidazole- and clarithromycin-resistant strains of H. pylori,
with minimal inhibitory concentration (MIC) values in the range of 4 to 32 mg/L. The dynamics of the decline in the bacterial counts at 2 X MIC appeared to be correlated with the lipophilicity of the drugs, suggesting different translocation efficiencies of DHPs across the bacterial membrane. Oral treatments with 100 mg/kg/day of marketed formulations of Nimodipine or Nitrendipine in combination with omeprazole significantly reduced the H. pylori gastric colonization in mice. The results presented here support a novel therapeutic solution for treatment of antibiotic-resistant H. pylori infections.
... Site-directed chemical modification of CA can be achieved by using thiol reactive compounds like as O-mesitylenesulfonylhydroxylamine (MSH) (Bernardes et al. 2008), maleimides (Kim et al. 2008), and alkenoic amides (Shiu et al. 2009). CA modification with most classes of inhibitors binds toward the metal center and generally substitutes hydroxide ion (Nishimori et al. 2008;Supuran 2008). Activators like amines, amino acids, and oligopeptides are generally bonded at the entrance of the active site cavity of CA enzyme. ...
Carbonic anhydrase modification (chemical and biological) is an attractive strategy for its diverse application to accelerate the absorption of CO2 from a flue gas with improved activity and stability. This article reports various possibilities of CA modification using metal–ligand homologous chemistry, cross-linking agents, and residue- and group-specific and genetic modifications, and assesses their role in carbon management. Chemically modified carbonic anhydrase is able to improve the absorption of carbon dioxide from a gas stream into mediation compounds with enhanced sequestration and mineral formation. Genetically modified CA polypeptide can also increase carbon dioxide conversion. Chemical modification of CA can be categorized in terms of (i) residue-specific modification (involves protein–ligand interaction in terms of substitution/addition) and group-specific modifications (based on the functional groups of the target CA). For every sustainable change, there should be no/limited toxic or immunological response. In this review, several CA modification pathways and biocompatibility rules are proposed as a theoretical support for emerging research in this area.
... are metalloproteins involved in several metabolic processes [8][9][10][11][12][13][14][15][16][17] either directly, by providing CO 2 /bicarbonate for carboxylating reactions [8,9,12,13], or indirectly, by modulating pH [13][14][15][16][17]. Indeed, CAs convert the neutral molecules CO 2 and water to a weak base (bicarbonate) and a strong acid (hydronium ion) with very high efficacy [8,13]. For this reason, in many organisms including bacteria, CAs are the main players in pH homeostasis and related physiologic processes, which are involved in the metabolism, survival and colonization of various niches in which these organisms thrive [18][19][20][21][22][23][24][25]. These processes were mainly investigated for pathogenic microorganisms, such as bacteria [9,13,16,17], protozoa [10,11,21,22] and fungi [17,26]. ...
The γ-carbonic anhydrases (CAs, EC 4.2.1.1) present in the Antarctic marine bacteria Pseudoalteromonas haloplanktis and Colwellia psychrerythraea, herein referred to as PhaCA and CpsCA, respectively, were investigated for their activation with a panel of 24 amino acids and amines. Both bacteria are considered Antarctic models for the investigation of photosynthetic and metabolic pathways in organisms adapted to live in cold seawater. PhaCA was much more sensitive to activation by these compounds compared to the genetically related enzyme CpsCA. The most effective PhaCA activators were d-Phe, l-/d-DOPA, l-Tyr and 2-pyridyl-methylamine, with the activation constant KA values of 0.72–3.27 µM. d-His, l-Trp, d-Tyr, histamine, dopamine, serotonin anddicarboxylic amino acids were also effective activators of PhaCA, with KA values of 6.48–9.85 µM. CpsCA was activated by d-Phe, d-DOPA, l-Trp, l-/d-Tyr, 4-amino-l-Phe, histamine, 2-pyridyl-methylamine and l-/d-Glu with KA values of 11.2–24.4 µM. The most effective CpsCA activator was l-DOPA (KA of 4.79 µM). Given that modulators of CAs from Antarctic bacteria have not been identified and investigated in detail for their metabolic roles to date, this research sheds some light on these poorly understood processes.
... Regard the HP, HP has two forms of CA, an alpha-type enzyme (HpαCA) and the Hp beta CA (HpβCA) (53,54). While HpαCA supports urease activity, HpβCA supports bacterial growth at acidic pH (54). ...
Helicobacter pylori (HP) is a facultative anaerobic bacterium. HP is a normal flora having immunomodulating properties. This bacterium is an example of a microorganism inducing gastric cancer. Its carcinogenicity depends on bacteria-host related factors. The proper understanding of the biology of HP inducing gastric cancer offers the potential strategy in the managing of HP rather than eradicating it. In this article, we try to summarize the biology of HP-induced gastric cancer and discuss the current pharmacological approach to treat and prevent its carcinogenicity.
... in various pathogenic organisms belonging to the bacteria, fungal or protozoan domains [11][12][13]. These enzymes effectively catalyze the reaction between CO 2 and water, with the formation of bicarbonate (HCO 3 − ) and protons (H + ), being among the very fast catalysts known in nature [14][15][16][17][18][19][20]. CAs are multifunctional enzymes which play central roles in various physiological, biochemical, and metabolic processes, such as acid-base homeostasis, respiratory gas exchange, electrolytes secretion, biosynthesis of urea, glucose, fatty acids, and carbamoyl phosphate, and also in the ionic transport, muscular contraction (in vertebrates), and photosynthesis (in plants and algae). ...
A newly described β-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic protozoan Entamoeba histolytica, EhiCA, was recently shown to possess a significant catalytic activity for the physiologic CO2 hydration reaction (kcat of 6.7 × 105 s−1 and a kcat/Km of 8.9 × 107 M−1 s−1). A panel of sulphonamides and one sulfamate, some of which are clinically used drugs, were investigated for their inhibitory properties against EhiCA. The best inhibitors detected in the study were 4-hydroxymethyl/ethyl-benzenesulfonamide (KIs of 36–89 nM), whereas some sulfanilyl-sulfonamides showed activities in the range of 285–331 nM. Acetazolamide, methazolamide, ethoxzolamide, and dichlorophenamide were less effective inhibitors (KIs of 509–845 nM) compared to other sulfonamides investigated here. As β-CAs are not present in vertebrates, the present study may be useful for detecting lead compounds for the design of more effective inhibitors with potential to develop anti-infectives with alternative mechanisms of action.
... The simple but significantly important hydration of CO 2 to HCO 3 − and H + makes CA highly critical for various steps of the bacterial life cycle including survival, invasion, and pathogenicity [45][46][47][48][49][50][51][52]. The highly negative impact of bacterial CAs inhibition on the bacteria life cycle has been already confirmed by various in vivo studies [53][54][55]. Therefore, biological end point of bacterial CAs induced by selective chemical inhibitors can be a potent strategy to defeat the antibiotic resistance of the pathogens. ...
Nowadays, antibiotic resistance has turned into one of the most important worldwide health problems. Biological end point of critical enzymes induced by potent inhibitors is recently being considered as a highly effective and popular strategy to defeat antibiotic-resistant pathogens. For instance, the simple but critical β-carbonic anhydrase has recently been in the center of attention for anti-pathogen drug discoveries. However, no β-carbonic anhydrase selective inhibitor has yet been developed. Available β-carbonic anhydrase inhibitors are also highly potent with regard to human carbonic anhydrases, leading to severe inevitable side effects in case of usage. Therefore, developing novel inhibitors with high selectivity against pathogenic β-carbonic anhydrases is of great essence. Herein, for the first time, we have conducted a proteochemometric study to explore the structural and the chemical aspects of the interactions governed by bacterial β-carbonic anhydrases and their inhibitors. We have found valuable information which can lead to designing novel inhibitors with better selectivity for bacterial β-carbonic anhydrases.
Aim: Development of dual-acting antibacterial agents containing Erlotinib, a recognized EGFR inhibitor used as an anticancer agent, with differently spaced benzenesulfonamide moieties known to bind and inhibit Helicobacter pylori carbonic anhydrase ( HpCA) or the antiviral Zidovudine. Methods & materials: Through rational design, ten derivatives were obtained via a straightforward synthesis including a click chemistry reaction. Inhibitory activity against a panel of pathogenic carbonic anhydrases and antibacterial susceptibility of H. pylori ATCC 43504 were assessed. Docking studies on α-carbonic anhydrase enzymes and EGFR were conducted to gain insight into the binding mode of these compounds. Results & conclusion: Some compounds proved to be strong inhibitors of HpCA and showed good anti- H. pylori activity. Computational studies on the targeted enzymes shed light on the interaction hotspots.
Introduction:
Four different genetic families of the enzyme carbonic anhydrase (CA, EC 4.2.1.1) are present in bacteria, α-, β-, γ- and ι-CAs. They play relevant functions related to CO2, HCO3-/H+ ions homeostasis, being involved in metabolic biosynthetic pathways, pH regulation, and represent virulence and survival factors for bacteria in various niches. Bacterial CAs started to be considered druggable targets in the last decade, as their inhibition impairs survival, growth, and virulence of these pathogens.
Areas covered:
Significant advances were registered in the last years for designing effective inhibitors of sulfonamide type for Helicobacter pylori α-CA, Neisseria gonorrhoeae α-CA, vacomycin-resistant enterococci (VRE) α- and γ-CAs, for which the in vivo validation has also been achieved. MIC-s in the range of 0.25-4.0 µg/mL for wild type and drug resistant N. gonorrhoeae strains, and of 0.007-2.0 µg/mL for VRE were observed for some 1,3,4-thiadiazole-2-sulfonamides, and acetazolamide was effective in gut decolonization from VRE.
Expert opinion:
Targeting bacterial CAs from other pathogens, among which Vibrio cholerae, Mycobacterium tuberculosis, Brucella suis, Salmonella enterica serovar Typhimurium, Legionella pneumophila, Porphyromonas gingivalis, Clostridium perfringens, Streptococcus mutans, Burkholderia pseudomallei, Francisella tularensis, Escherichia coli, Mammaliicoccus (Staphylococcus) sciuri, Pseudomonas aeruginosa, may lead to novel antibacterials devoid of drug resistance problems.
Advances in the carbonic anhydrase (CA, EC 4.2.1.1) research over the last three decades are presented, with an emphasis on the deciphering of the activation mechanism, the development of isoform-selective inhibitors/ activators by the tail approach and their applications in the management of obesity, hypoxic tumors, neurological conditions, and as antiinfectives.
Increase in concentration of greenhouse gases is one of the major factors of climate change and among them contribution of carbon dioxide is more than other gases. Increase in temperature, warming of oceans, rise in sea level, shirking ice-sheaths, glacial retreat, ocean acidification are the consequences of increase in CO2 level. Human beings are also likely to be affected by various
diseases at times. Globally much efforts have been made to reduce CO2 emission, but they are highly expensive and energy
intensive. Carbonic anhydrase (CA) that can catalyze the reversible hydration and dehydration of CO2 and HCO3
− respectively has arrived a better and ecofriendly enzymatic method for CO2 removal. Thus, one of the most efficient approaches is the use of CA as a biocatalyst in the biosequestration of carbon. For industrial application of CA in biomimetic carbon sequestration, it needs to possess two essential properties, viz., thermostability and alkali stability, that can be achieved by approaches such as protein engineering, whole cell biocatalysis and immobilization. Enzyme immobilization methods have opened a new path for industrial scale applications by effective enzymatic recovery, reusability, and long-term operating stability. The various methods of immobilization with their merits and limitations along with ongoing research work in this field has been addressed here. The relevance of bioinformatics is being realized in the era of genomics, assisting in genome wide identification, characterization of putative gene families of different enzymes for diverse industrial applications. This analysis for industrially important enzyme has been done by authors to improve the catalytic efficiency, thermostability and structural stability. This review also gives a structural overview of different classes of CA discovered till date, and their evolutionary relationships has been studied using multiple sequence alignment (MSA).
Neisseria gonorrhoeae is a high-priority pathogen of concern due to the growing prevalence of resistance development against approved antibiotics. Herein, we report the anti-gonococcal activity of ethoxzolamide, the FDA-approved human carbonic anhydrase inhibitor. Ethoxzolamide displayed an MIC50, against a panel of N. gonorrhoeae isolates, of 0.125 µg/mL, 16-fold more potent than acetazolamide, although both molecules exhibited almost similar potency against the gonococcal carbonic anhydrase enzyme (NgCA) in vitro. Acetazolamide displayed an inhibition constant (Ki) versus NgCA of 74 nM, while Ethoxzolamide’s Ki was estimated to 94 nM. Therefore, the increased anti-gonococcal potency of ethoxzolamide was attributed to its increased permeability in N. gonorrhoeae as compared to that of acetazolamide. Both drugs demonstrated bacteriostatic activity against N. gonorrhoeae, exhibited post-antibiotic effects up to 10 hours, and resistance was not observed against both. Taken together, these results indicate that acetazolamide and ethoxzolamide warrant further investigation for translation into effective anti-N. gonorrhoeae agents.
Introduction
The clinically licensed drugs used as antibiotics prevent the microbial growth interfering with the biosynthesis of proteins, nucleic acids, microorganism wall biosynthesis or wall permeability, and microbial metabolic pathways. A serious, emerging problem is the arisen of extensive drug resistance afflicting most countries worldwide.
Areas covered
An exciting approach to fight drug resistance is the identification of essential enzymes encoded by pathogen genomes. Inhibition of such enzymes may impair microbial growth or virulence due to interference with crucial metabolic processes. Genome exploration of pathogenic and non-pathogenic microorganisms has revealed carbonic anhydrases (CAs, EC 4.2.1.1) as possible antibacterial targets.
Expert opinion
Balancing the equilibrium between CO2 and HCO3 ⁻ is essential for microbial metabolism and is regulated by at least four classes of CAs. Classical CA inhibitors (CAIs) such ethoxzolamide were shown to kill the gastric pathogen Helicobacter pylori in vitro, whereas acetazolamide and some of its more lipophilic derivatives were shown to be effective against vancomycin-resistant Enterococcus spp., with MICs in the range of 0.007 – 2 µg/mL, better than linezolid, the only clinically used agent available to date. Such results reinforce the rationale of considering existing and newly designed CAIs as antibacterials with an alternative mechanism of action.
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes involved in biosynthetic processes, transport, supply, and balance of CO2/HCO3⁻ into the cell. In Bacteria, CAs avoid the depletion of the dissolved CO2/HCO3⁻ from the cell, providing them to the central metabolism that is compromised without the CA activity. The involvement of CAs in the survival, pathogenicity, and virulence of several bacterial pathogenic species is recent. Here, we report the kinetic properties of the recombinant γ-CA (EcoCAγ) encoded in the genome of Escherichia coli. EcoCAγ is an excellent catalyst for the physiological CO2 hydration reaction to bicarbonate and protons, with a kcat of 5.7 × 10⁵ s⁻¹ and kcat/KM of 6.9 × 10⁶ M⁻¹ s⁻¹. The EcoCAγ inhibition profile with a broad series of known CA inhibitors, the substituted benzene-sulphonamides, and clinically licenced drugs was explored. Benzolamide showed a KI lower than 100 nM. Our study reinforces the hypothesis that the synthesis of new drugs capable of interfering selectively with the bacterial CA activity, avoiding the inhibition of the human α -CAs, is achievable and may lead to novel antibacterials.
Vancomycin-resistant enterococci (VRE) are the second leading cause of hospital-acquired infections (HAIs) attributed to a drug-resistant bacterium in the United States and resistance to the frontline treatments is well documented. To combat VRE, we have repurposed the FDA-approved carbonic anhydrase drug acetazolamide to design potent anti-enterococcal agents. Through structure-activity relationship optimization we have arrived at two leads possessing improved potency against clinical VRE strains from MIC = 2 μg/mL (acetazolamide) to MIC = 0.007 μg/mL (22) and 1 μg/mL (26). Physicochemical properties were modified to design leads that either have high oral bioavailability to treat systemic infections or low intestinal permeability to treat VRE infections in the gastrointestinal tract. Our data suggests the intracellular target for the molecules are putative α-carbonic and γ-carbonic anhydrases and homology modeling and molecular dynamics simulations were performed. Together, this study presents potential anti-VRE therapeutic options to provide alternatives for problematic VRE infections.
Spermine and spermidine polyamines are investigated as a new class inhibitors of different carbonic anhydrase (CA) isoforms using DFT calculations. Our results indicate these two polyamines, inhibited the human carbonic anhydrase with very different inhibition profiles compared to other inhibitors. According to calculated results, studied polyamines are anchored to the non-protein zinc ligand (hydroxyl ion) by means of a hydrogen bond of 2.6 Å involving one of terminal ammonium group and an intermediate complex is formed, [(his)3Zn(II)(OH)/polyamine], which is in good agreement with experimental data. By transferring a proton from the terminal ammonium group of polyamine inhibitor to the oxygen atom of the hydroxyl group, the active form of the CA enzyme converted to the inactive form. Finally, the HOMO-LUMO and AIM analysis have been done to understand the details of interaction between studied polyamines and CA active center in intermediate complex in water phase.
The carbonic anhydrases (CAs, EC 4.2.1.1) catalyse a simple but physiologically crucial reversible reaction, the carbon dioxide hydration with the production of bicarbonate and protons. In the last years, and especially, to the rapid emergence of the bacterial antibiotic resistance that is occurring worldwide, the understanding of the function of bacterial CAs has increased significantly. Recently, a new CA-class (ι-CA) was discovered in the marine diatom T. pseudonana. It has been reported that bacterial genomes may contain genes with relevant homology to the diatom ι-class CA. Still, the catalytic activity of the enzyme encoded by the gene was not investigated. Thus, herein, for the first time, we cloned, expressed, and purified the recombinant bacterial ι-CA (acronym BteCAι) identified in the genome of Burkholderia territorii. The recombinant BteCAι resulted in a good catalyst for the hydration of CO2 to bicarbonate and protons, with a kcat of 3.0 × 10⁵ s ⁻¹ and kcat/KM of 3.9 × 10⁷ M ⁻¹ s ⁻¹, and is also sensitive to inhibition by the sulphonamide acetazolamide. Furthermore, with the aid of the protonography, it has been demonstrated that BteCAι can be present as a dimer. This result is corroborated by the construction of a molecular model of BteCAι, which showed that the enzyme is formed by two equivalent monomers having a structure similar to a butterfly.
Introduction: The spacious active site cavity of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) shows a great versatility for a variety of binding modes for modulators of activity, inhibitors, and activators, some of which are clinically used drugs.
Areas covered: There are at least four well-documented CA inhibition mechanisms and the same number of binding modes for CA inhibitors (CAIs), one of which superposes with the binding of activators (CAAs). They include (i) coordination to the catalytic metal ion; (ii) anchoring to the water molecule coordinated to the metal ion; (iii) occlusion of the active site entrance; and (iv) binding outside the active site. A large number of chemical classes of CAIs show these binding modes explored in detail by kinetic, crystallographic, and other techniques. The tail approach was applied to all of them and allowed many classes of highly isoform-selective inhibitors. This is the subject of our review.
Expert opinion: All active site regions of CAs accommodate inhibitors to bind, which is reflected in very different inhibition profiles for such compounds and the possibility to design drugs with effective action and new applications, such as for the management of hypoxic tumors, neuropathic pain, cerebral ischemia, arthritis, and degenerative disorders.
The genome of Mycobacterium tuberculosis (Mtb) encodes three β-carbonic anhydrases (CAs, EC 4.2.1.1) that are crucial for the life cycle of the bacterium. The Mtb β-CAs have been cloned and characterized, and the catalytic activities of the enzymes have been studied. The crystal structures of two of the enzymes have been resolved. In vitro inhibition studies have been conducted using different classes of carbonic anhydrase inhibitors (CAIs). In vivo inhibition studies of pathogenic bacteria containing β-CAs showed that β-CA inhibitors effectively inhibited the growth of pathogenic bacteria. The in vitro and in vivo studies clearly demonstrated that β-CAs of not only mycobacterial species, but also other pathogenic bacteria, can be targeted for developing novel antimycobacterial agents for treating tuberculosis and other microbial infections that are resistant to existing drugs. In this review, we present the molecular and structural data on three β-CAs of Mtb that will give us better insights into the roles of these enzymes in pathogenic bacterial species. We also present data from both in vitro inhibition studies using different classes of chemical compounds and in vivo inhibition studies focusing on M. marinum, a model organism and close relative of Mtb.
Ethoxzolamide (EZA), acetazolamide, and methazolamide are clinically used sulphonamide drugs designed to treat non-bacteria-related illnesses (e.g. glaucoma), but they also show antimicrobial activity against the gastric pathogen Helicobacter pylori. EZA showed the highest activity, and was effective against clinical isolates resistant to metronidazole, clarithromycin, and/or amoxicillin, suggesting that EZA kills H. pylori via mechanisms different from that of these antibiotics. The frequency of single-step spontaneous resistance acquisition by H. pylori was less than 5 × 10⁻⁹, showing that resistance to EZA does not develop easily. Resistance was associated with mutations in three genes, including the one that encodes undecaprenyl pyrophosphate synthase, a known target of sulphonamides. The data indicate that EZA impacts multiple targets in killing H. pylori. Our findings suggest that developing the approved anti-glaucoma drug EZA into a more effective anti-H. pylori agent may offer a faster and cost-effective route towards new antimicrobials with a novel mechanism of action.
The cloning, purification, and initial characterization of the β-carbonic anhydrase (CA, EC 4.2.1.1) from the genome of the opportunistic pathogen Malassezia restricta (MreCA), which a fungus involved in dandruff and seborrheic dermatitis (SD), is reported. MreCA is a protein consisting of 230 amino acid residues and shows high catalytic activity for the hydration of CO2 into bicarbonate and protons, with the following kinetic parameters: kcat of 1.06 × 106 s−1 and kcat/KM of 1.07 × 108 M−1 s−1. It is also sensitive to inhibition by the sulfonamide acetazolamide (KI of 50.7 nM). Phylogenetically, MreCA and other CAs from various Malassezia species seem to be on a different branch, distinct from that of other β-CAs found in fungi, such as Candida spp., Saccharomyces cerevisiae, Aspergillus fumigatus, and Sordaria macrospora, with only Cryptococcus neoformans and Ustilago maydis enzymes clustering near MreCA. The further characterization of this enzyme and the identification of inhibitors that may interfere with its life cycle might constitute new strategies for fighting dandruff and SD.
Carbonic anhydrase is a metalloprotein, an enzyme with strong inhibition in antibacterial treatment. This study presents QSAR modeling for a series of 41 chemical compounds, 40 sulfonamides and one sulfamate, including 13 clinically tested drugs as carbonic anhydrase inhibitors based on the Monte Carlo optimization with molecular descriptors based on the SMILES notation and local invariants of the molecular graph, and field 3D based methods. Conformation independent QSAR models were developed for three random splits and a 3D QSAR model for one random split into the training and test sets. The statistical quality of the developed models, including robustness and predictability, was tested using various statistical approaches and the results that were obtained were very good. An excellent correlation between the results from the conformation independent and the 3D QSAR model was obtained. A novel statistical metric known as the index of ideality of correlation was used for the final assessment of the model, and the obtained results were good. Molecular fragments responsible for the increases and decreases of a studied activity were defined and further used for the computer-aided design of new compounds as potential carbonic anhydrase inhibitors. Molecular docking was applied for the final assessment of the developed QSAR model and designed inhibitors, and an excellent correlation between the results from QSAR modeling and molecular docking studies was obtained.
Communicated by Ramaswamy H. Sarma
Carbonic anhydrases (CAs, EC 4.2.1.1) are a superfamily of ubiquitous metalloenzymes present in all living organisms on the planet. They are classified into seven genetically distinct families and catalyse the hydration reaction of carbon dioxide to bicarbonate and protons, as well as the opposite reaction. CAs were proposed to be used for biotechnological applications, such as the post-combustion carbon capture processes. In this context, there is a great interest in searching CAs with robust chemical and physical properties. Here, we describe the enhancement of thermostability of the α-CA from Sulfurihydrogenibium yellowstonense (SspCA) by using the anchoring-and-self-labelling-protein-tag system (ASLtag). The anchored chimeric H⁵-SspCA was active for the CO2 hydration reaction and its thermostability increased when the cells were heated for a prolonged period at high temperatures (e.g. 70 °C). The ASLtag can be considered as a useful method for enhancing the thermostability of a protein useful for biotechnological applications, which often need harsh operating conditions.
Famotidine, an antiulcer drug belonging to the H2 antagonists class of pharmacological agents, was recently shown to potently inhibit human (h) and bacterial carbonic anhydrases (CAs, EC 4.2.1.1). We investigated the inhibitory effects of famotidine against all classes of CAs from the pathogenic bacteria Vibrio cholerae, Burkholderia pseudomallei and Mycobacterium tuberculosis Rv3273 β-CA, as well as the CAs from the nonpathogenic bacteria/cyanobacteria Sulfurihydrogenibium yellowstonensis, S. azorense, Pseudoalteromonas haloplanktis, Colwellia psychrerythraea and Nostoc commune. The δ- and ζ-CAs from the diatom Thalassiosira weissflogii, the fungal enzymes from Cryptococcus neoformans, Candida glabrata and Malassezia globosa, as well as the protozoan enzymes from Trypanosoma cruzi and Plasmodium falciparum, were also investigated. Anopheles gambiae β-CA was also investigated. All these enzymes were effectively inhibited by famotidine, with affinities between the low nanomolar to the micromolar range. The best inhibition was observed against C. glabrata β-CA and TweCAζ, with KIs ranging between 13.6 and 22.1 nM.
Carbonic anhydrases (CAs) are ubiquitous metalloenzymes, which started to be investigated in detail in pathogenic, as well as non-pathogenic species since their pivotal role is to accelerate the physiological CO2 hydration/dehydration reaction significantly. Here, we propose the marine unicellular diatom Phaeodactylum tricornutum as a model organism for testing the membrane penetrability of CA inhibitors (CAIs). Seven inhibitors belonging to the sulphonamide type and possessing a diverse scaffold have been explored for their in vitro inhibition of the whole diatom CAs and the in vivo inhibitory effect on the growth of P. tricornutum. Interesting, inhibition of growth was observed, in vivo, demonstrating that this diatom is a good model for testing the cell wall penetrability of this class of pharmacological agents. Considering that many pathogens are difficult and dangerous to grow in the laboratory, the growth inhibition of P. tricornutum with different such CAIs may be subsequently used to design inhibition studies of CAs from pathogenic organisms.
Introduction: In all living species, pH regulation is a tightly controlled process, with a plethora of proteins involved in its regulation. These include sodium-proton exchangers, carbonic anhydrases, anion exchangers, bicarbonate transporters/cotransporters, H⁺-ATPases, and monocarboxylate transporters. All of them play crucial roles in acid-base balancing, both in eukaryotic as well as in prokaryotic organisms, making them interesting drug targets for the management of pathological events (in)directly involved in pH regulation.
Areas covered: Interfering with pH regulation for the treatment of tumors and microbial infections is the main focus of this review, with particular attention paid to inhibitors targeting the above-mentioned proteins. The latest advances in each field id reviewed.
Expert opinion: Interfering with the pH regulation of tumor cells is a validated approach to tackle primary tumors and metastases growth. Carbonic anhydrases are the most investigated proteins of those aforementioned, with several inhibitors in clinical development. Recent advances in the characterization of proteins involved in pH homeostasis of various pathogens evidenced their crucial role in the survival and virulence of bacterial, fungal, and protozoan microorganisms. Some encouraging results shed light on the possibility to target such proteins for obtaining new anti-infectives, overcoming the extensive drug resistance problems of clinically used drugs.
The genome of Helicobacter pylori encodes for carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α- and β-CA classes, which together with urease, have a pivotal role in the acid acclimation of the microorganism within the human stomach. Recently, in the exoproteome of H. pylori, a CA with no indication of the corresponding class was identified. Here, using the protonography and the mass spectrometry, a CA belonging to the α-class was detected in the outer membrane vesicles (OMVs) generated by planktonic and biofilm phenotypes of four H. pylori strains. The amount of this metalloenzyme was higher in the planktonic OMVs (pOMVs) than in the biofilm OMVs (bOMVs). Furthermore, the content of α-CA increases over time in the pOMVs. The identification of the α-CA in pOMVs and bOMVs might shed new light on the role of this enzyme in the colonization, survival, persistence, and pathogenesis of H. pylori.
A series of benzenesulfonamides incorporating selenazoles with diverse substitution patterns were investigated as inhibitors of six bacterial carbonic anhydrases (CAs, EC 4.2.1.1) from bacterial pathogens, such as Helicobacter pylori (hpCAα was the investigated enzyme), Vibrio cholerae (all the three CAs from this pathogen were considered, VchCAα, VchCAβ and VchCAγ) and Burkholderia pseudomallei (with its two CAs, BpsCAβ and BpsCAγ). All these sulfonamides were effective CA inhibitors, with potencies in the low micromolar or submicromolar range, making them attractive as lead compounds for designing antibacterials with a novel mechanism of action, which could counteract the extensive resistance problem observed with many clinically used antibiotics.
X-ray and neutron crystallography are powerful techniques utilized to study the structures of biomolecules. Visualization of enzymes in complex with substrate/product and the capture of intermediate states can be related to activity to facilitate understanding of the catalytic mechanism. Subsequent analysis of small molecule binding within the enzyme active site provides insight into mechanisms of inhibition, supporting the design of novel inhibitors using a structure-guided approach. The first X-ray crystal structures were determined for small, ubiquitous enzymes such as carbonic anhydrase (CA). CAs are a family of zinc metalloenzymes that catalyze the hydration of CO 2 , producing HCO3- and a proton. The CA structure and ping-pong mechanism have been extensively studied and are well understood. Though the function of CA plays an important role in a variety of physiological functions, CA has also been associated with diseases such as glaucoma, edema, epilepsy, obesity, and cancer and is therefore recognized as a drug target. In this review, a brief history of crystallography and its impact on CA research is discussed.
Pseudomonas aeruginosa (P. aeruginosa) is a gram‐negative facultative anaerobe belonging to the Pseudomonadaceae family. It is a multi‐drug resistant opportunistic human pathogen and a common cause of life‐threatening nosocomial infections, and is a key bacterial agent in cystic fibrosis and endocarditis diseases. The bacterium exhibits intrinsic resistance to most antibacterial agents, including aminoglycosides and quinolones. Hence, the identification of new drug targets for P. aeruginosa are ongoing. PsCA3 is a β‐class carbonic anhydrase (β‐CA) catalyzing the reversible hydration of carbon dioxide to bicarbonate and represents a new class of antimicrobial target. Previously, inhibitor‐screening studies of psCA3 have shown a series of small anions including sulfamide (SFN), imidazole (IMD), and 4‐methyl imidazole (4MI) and thiocyanate (SCN) inhibit the enzyme with efficiencies in the micro‐ to millimolar range. Here, the X‐ray crystal structures of these inhibitors in complex with psCA3 are presented and compared to human CA II. This structural survey into the binding modes of small anions forms the foundation for the development of inhibitors against β‐CAs and more selective inhibitors against P. aeruginosa.
Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread metalloenzymes used by living organisms to accelerate the CO2 hydration/dehydration reaction at rates dramatically high compared to the uncatalyzed reaction. These enzymes have different isoforms and homologues and can be found in the form of cytoplasmic, secreted or membrane-bound proteins. CAs play a role in numerous physiological processes including biomineralization and symbiosis, as is the case in reef-building corals. Previously, molecular and biochemical data have been obtained at the molecular level in the branching coral Stylophora pistillata for two coral isoforms which differ significantly in their catalytic activity and susceptibility to inhibition with anions and sulfonamides. More recently it has been determined that the genome of S. pistillata encodes for 16 CAs. Here, we cloned, expressed, purified and characterized a novel α-CA, named SpiCA3, which is cytoplasmic and ubiquitously expressed in all the cell layers including the calcifying cells. SpiCA3 is the most effective CA among the coral isoforms investigated and the most efficient catalyst known up to date in Metazoa. We also investigated the inhibition profiles of SpiCA3 and compared it with those obtained for the two other isoforms in the presence of inorganic anions and other small molecules known to interfere with metalloenzymes. These results suggest that S. pistillata has adapted its CA isoforms to achieve the physiological functions in different physicochemical microenvironments.
Introduction: The hydration/dehydration of CO2 catalyzed by carbonic anhydrases (CAs, EC 4.2.1.1) is a crucial physiological reaction for the survival of all living organisms because it is connected with numerous biosynthetic and biochemical pathways requiring CO2 or HCO3⁻, such as respiration, photosynthesis, carboxylation reactions, pH homeostasis, secretion of electrolytes, transport of CO2 and bicarbonate, etc.
Areas Covered: The bacterial genome encodes CAs belonging to the α-, β-, and γ-CA classes able to ensure the survival and/or satisfying the metabolic needs of the bacteria, as demonstrated by in vivo and in vitro experiments. The discovery of new anti-infectives that target new bacterial pathways, such as those involving CAs, may lead to effective therapies against diseases subject to the antibiotic resistance. This aspect is important in pharmaceutical and biomedical research but received little attention till recently.
Expert Opinion: An overview of the potential use of CAs in biomedical applications, as drug targets, bioindicators, and within artificial organs is presented. The discovery of thermostable bacterial CAs allowed the use of CAs in biotechnological applications, but patents related to the use of bacterial CAs in the development of pharmacological agents are scarce.
Carbonic anhydrases catalyze the reversible hydration of CO(2) and are ubiquitous in highly evolved eukaryotes. The recent identification of a third class of carbonic anhydrase (gamma class) in a methanoarchaeon and our present finding that the beta class also extends into thermophilic species from the Archaea domain led us to initiate a systematic search for these enzymes in metabolically and phylogenetically diverse prokaryotes. Here we show that carbonic anhydrase is widespread in the Archaea and Bacteria domains, and is an ancient enzyme. The occurrence in chemolithoautotrophic species occupying deep branches of the universal phylogenetic tree suggests a role for this enzyme in the proposed autotrophic origin of life. The presence of the beta and gamma classes in metabolically diverse species spanning the Archaea and Bacteria domains demonstrates that carbonic anhydrases have a far more extensive and fundamental role in prokaryotic biology than previously recognized.
Helicobacter pylori, strain 26695, has a circular genome of 1,667,867 base pairs and 1,590 predicted coding sequences. Sequence analysis indicates that H. pylori has well-developed systems for motility, for scavenging iron, and for DNA restriction and modification. Many putative adhesins, lipoproteins and other outer membrane proteins were identified, underscoring the potential complexity of host-pathogen interaction. Based on the large number of sequence-related genes encoding outer membrane proteins and the presence of homopolymeric tracts and dinucleotide repeats in coding sequences, H. pylori, like several other mucosal pathogens, probably uses recombination and slipped-strand mispairing within repeats as mechanisms for antigenic variation and adaptive evolution. Consistent with its restricted niche, H. pylori has a few regulatory networks, and a limited metabolic repertoire and biosynthetic capacity. Its survival in acid conditions depends, in part, on its ability to establish a positive inside-membrane potential in low pH.
Helicobacter pylori, one of the most common bacterial pathogens of humans, colonizes the gastric mucosa, where it appears to persist throughout the host's life unless the patient is treated. Colonization induces chronic gastric inflammation which can progress to a variety of diseases, ranging in severity from superficial gastritis and peptic ulcer to gastric cancer and mucosal-associated lymphoma. Strain-specific genetic diversity has been proposed to be involved in the organism's ability to cause different diseases or even be beneficial to the infected host and to participate in the lifelong chronicity of infection. Here we compare the complete genomic sequences of two unrelated H. pylori isolates. This is, to our knowledge, the first such genomic comparison. H. pylori was believed to exhibit a large degree of genomic and allelic diversity, but we find that the overall genomic organization, gene order and predicted proteomes (sets of proteins encoded by the genomes) of the two strains are quite similar. Between 6 to 7% of the genes are specific to each strain, with almost half of these genes being clustered in a single hypervariable region.
We have determined the structure of the beta-carbonic anhydrase from the dicotyledonous plant Pisum sativum at 1.93 A resolution, using a combination of multiple anomalous scattering off the active site zinc ion and non-crystallographic symmetry averaging. The mol- ecule assembles as an octamer with a novel dimer of dimers of dimers arrangement. Two distinct patterns of conservation of active site residues are observed, implying two potentially mechanistically distinct classes of beta-carbonic anhydrases. The active site is located at the interface between two monomers, with Cys160, His220 and Cys223 binding the catalytic zinc ion and residues Asp162 (oriented by Arg164), Gly224, Gln151, Val184, Phe179 and Tyr205 interacting with the substrate analogue, acetic acid. The substrate binding groups have a one to one correspondence with the functional groups in the alpha-carbonic anhydrase active site, with the corresponding residues being closely superimposable by a mirror plane. Therefore, despite differing folds, alpha- and beta-carbonic anhydrase have converged upon a very similar active site design and are likely to share a common mechanism.
The structure of the “cab”-type β class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) has been determined to 2.1-Å resolution using the multiwavelength anomalous diffraction phasing technique. Cab exists
as a dimer with a subunit fold similar to that observed in “plant”-type β class carbonic anhydrases. The active site zinc
is coordinated by protein ligands Cys32, His87, and Cys90, with the tetrahedral coordination completed by a water molecule. The major difference between plant- and cab-type β class
carbonic anhydrases is in the organization of the hydrophobic pocket. The structure reveals a Hepes buffer molecule bound
8 Å away from the active site zinc, which suggests a possible proton transfer pathway from the active site to the solvent.
H. pylori is one of the most common bacterial infections in human beings, and its discovery 20 years ago altered the diagnosis and treatment of gastroduodenal disease. This review considers current knowledge about the epidemiology and transmission of H. pylori, as well as the role of this infectious agent in the pathogenesis of upper gastrointestinal tract disease. Diagnostic approaches, indications for therapy, and measures of therapeutic efficacy are reviewed.
The role of the periplasmic α-carbonic anhydrase (α-CA) (HP1186) in acid acclimation of Helicobacter pylori was investigated. Urease and urea influx through UreI have been shown to be essential for gastric colonization and for acid
survival in vitro. Intrabacterial urease generation of NH3 has a major role in regulation of periplasmic pH and inner membrane potential under acidic conditions, allowing adequate
bioenergetics for survival and growth. Since α-CA catalyzes the conversion of CO2 to HCO3−, the role of CO2 in periplasmic buffering was studied using an α-CA deletion mutant and the CA inhibitor acetazolamide. Western analysis confirmed
that α-CA was bound to the inner membrane. Immunoblots and PCR confirmed the absence of the enzyme and the gene in the α-CA
knockout. In the mutant or in the presence of acetazolamide, there was an ∼3 log10 decrease in acid survival. In acid, absence of α-CA activity decreased membrane integrity, as observed using membrane-permeant
and -impermeant fluorescent DNA dyes. The increase in membrane potential and cytoplasmic buffering following urea addition
to wild-type organisms in acid was absent in the α-CA knockout mutant and in the presence of acetazolamide, although UreI
and urease remained fully functional. At low pH, the elevation of cytoplasmic and periplasmic pH with urea was abolished in
the absence of α-CA activity. Hence, buffering of the periplasm to a pH consistent with viability depends not only on NH3 efflux from the cytoplasm but also on the conversion of CO2, produced by urease, to HCO3− by the periplasmic α-CA.
Helicobacter pylori produces acute superficial gastritis in nearly all of its human hosts. However, a subset of individuals develops chronic atrophic gastritis (ChAG), a condition characterized in part by diminished numbers of acid-producing parietal cells and increased risk for development of gastric adenocarcinoma. Previously, we used a gnotobiotic transgenic mouse model with an engineered ablation of parietal cells to show that loss of parietal cells provides an opportunity for a H. pylori isolate from a patient with ChAG (HPAG1) to bind to, enter, and persist within gastric stem cells. This finding raises the question of how ChAG influences H. pylori genome evolution, physiology, and tumorigenesis. Here we describe the 1,596,366-bp HPAG1 genome. Custom HPAG1 Affymetrix GeneChips, representing 99.6% of its predicted ORFs, were used for whole-genome genotyping of additional H. pylori ChAG isolates obtained from Swedish patients enrolled in a case-control study of gastric cancer, as well as ChAG- and cancer-associated isolates from an individual who progressed from ChAG to gastric adenocarcinoma. The results reveal a shared gene signature among ChAG strains, as well as genes that may have been lost or gained during progression to adenocarcinoma. Whole-genome transcriptional profiling of HPAG1’s response to acid during in vitro growth indicates that genes encoding components of metal uptake and utilization pathways, outer membrane proteins, and virulence factors are among those associated with H. pylori’s adaptation to ChAG.
• acid regulation
• comparative microbial genomics
• ecogenomics
• functional genomics
• gastric cancer
The cell division-related gene A (cdrA) of Helicobacter pylori is dispensable in vivo and unique in having a repressive role on cell division and long-term survival. To clarify its role, comparisons of the wildtype HPK5 and isogenic cdrA-disrupted mutant HPKT510 were examined by ultrastructural morphology, PBP profiles, and susceptibility to P-lactam antibiotics during long-term cultivation. Ultrastructural analyses revealed that the shorter rods of HPKT510 had a slightly wider periplasmic space between the inner and the outer membrane than those of HPK5. Cell division of HPKT510 cells was complete even under high-salt conditions in which HPK5 cells became filamentous due to inhibition of division. The filamentous HPK5 cells constructed an inner membrane without a cell wall at the presumed division site. After 4 days of cultivation (the late stationary phase), most of the HPK5 cells turned into ghosts and aggregates, while some of the HPKT510 cells remained as curved rods, which coincided with the results of cell viability. HPKT510 cells became resistant to ampicillin killing compared to HPK5 cells, although their minimum inhibitory concentrations (MICs) and PBP profiles were not significantly different. These results suggest that the cdrA product represses cell division via inhibiting cell wall synthesis at division site. During infection in both mice and humans, inactivation of cdrA eventually gains biological aspects such as increased viability, long-term survival and tolerance to antibiotics and high-salt condition, which might enhance a persistent infection.
The purpose of the present review was to determine objectively the optimal treatment for the eradication of H. pylori amongst the currently used regimens. A comprehensive literature search provided a data-base relating to the following treatments: dual therapy with an anti-secretory drug plus either amoxycillin or clarithromycin; standard triple therapy, with or without additional anti-secretory drugs; proton pump inhibitor triple therapy; and H2-receptor antagonist triple therapy. Emphasis was placed on intention-to-treat analyses of eradication rates using all of the available evidence. The criteria used to select the optimal treatment were efficacy (eradication rates), frequency of side-effects, simplicity of the regimen (number of tablets per day and duration of treatment) and cost. The analysis showed that proton pump inhibitor triple therapy (that is, a proton pump inhibitor plus any two of amoxycillin, clarithromycin or a nitroimidazole) was the preferred treatment for the eradication of H. pylori. In particular, the 1-week, low-dose regimen with omeprazole plus clarithromycin plus tinidazole produced the highest eradication rates (> 90%) with the lowest frequency of side-effects and at only modest cost.
The relative role of internal urease for acid protection of Helicobacter pylori is unknown. The aim of this study was to determine the comparative importance of internal and external urease under acidic conditions.
The pH optimum and measured Michaelis constant for urea of external urease and urease in intact bacteria at different medium pH (pHout) were measured using 14CO2 release from 14C-urea. The effect of urea on membrane potential and bacterial cytoplasmic pH was measured at different fixed pHout. 35S-methionine labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled proteins in the organism and medium measured protein synthesis at different pHout and mechanisms of urease externalization.
External urease had activity between pH 5.0 and 8.5 and internal urease between pHout 2.5 and 6.5, and its Michaelis constant at pHout 7.5 was 300 mmol/L but at pHout 4.5 was 0.5 mmol/L, similar to free urease. The addition of 5 mmol/L urea to bacteria at fixed pHout from 3.0 to 6.0 elevated potential to about -105 mV and periplasmic pH to about pH 6.2. Protein synthesis occurred mainly between pH 6.5 and 8.0, and urease activity resulted in increased protein synthesis at acidic pH. The labeling pattern of intrabacterial and released protein was similar.
Intracellular urease activity is regulated by external pH, defends against gastric acidity by increasing periplasmic pH and membrane potential, and stimulates protein synthesis at acidic pH. External urease is produced mostly by cell lysis.
Carbonic anhydrase (CA) (EC 4.2.1.1) enzymes catalyze the reversible hydration of CO2, a reaction that is important in many physiological processes. We have cloned and sequenced a full-length cDNA encoding an intracellular beta-CA from the unicellular green alga Coccomyxa. Nucleotide sequence data show that the isolated cDNA contains an open reading frame encoding a polypeptide of 227 amino acids. The predicted polypeptide is similar to beta-type CAs from Escherichia coli and higher plants, with an identity of 26% to 30%. The Coccomyxa cDNA was overexpressed in E. coli, and the enzyme was purified and biochemically characterized. The mature protein is a homotetramer with an estimated molecular mass of 100 kD. The CO2-hydration activity of the Coccomyxa enzyme is comparable with that of the pea homolog. However, the activity of Coccomyxa CA is largely insensitive to oxidative conditions, in contrast to similar enzymes from most higher plants. Fractionation studies further showed that Coccomyxa CA is extrachloroplastic.
Although the currently most effective treatment regimens cure about 90% of infections, 10% of patients remain Helicobacter pylori positive. Several factors contribute to treatment failure. These include patient compliance, bacterial resistance to antibiotics, and treatment related issues. Treatment failure leads to the development of bacterial resistance to metronidazole and clarithromycin. Retreatment can be undertaken after considering several different strategies: to repeat the same regimen with full doses of medications and a longer treatment duration, or to choose different regimens to avoid the antibiotic previously used, or to switch to proton pump inhibitor (PPI) based quadruple therapy or ranitidine bismuth citrate (RBC) based triple therapy. In principle, full doses and longer treatment durations are advisable. As retreatment is always difficult, choosing the best available first line treatment regimen is still the best "rescue" treatment.
Carbonic anhydrases catalyze the reversible hydration of CO(2) [CO(2)+H(2)Oright harpoon over left harpoon HCO(3)(-)+H(+)]. Since the discovery of this zinc (Zn) metalloenzyme in erythrocytes over 65 years ago, carbonic anhydrase has not only been found in virtually all mammalian tissues but is also abundant in plants and green unicellular algae. The enzyme is important to many eukaryotic physiological processes such as respiration, CO(2) transport and photosynthesis. Although ubiquitous in highly evolved organisms from the Eukarya domain, the enzyme has received scant attention in prokaryotes from the Bacteria and Archaea domains and has been purified from only five species since it was first identified in Neisseria sicca in 1963. Recent work has shown that carbonic anhydrase is widespread in metabolically diverse species from both the Archaea and Bacteria domains indicating that the enzyme has a more extensive and fundamental role in prokaryotic biology than previously recognized. A remarkable feature of carbonic anhydrase is the existence of three distinct classes (designated alpha, beta and gamma) that have no significant sequence identity and were invented independently. Thus, the carbonic anhydrase classes are excellent examples of convergent evolution of catalytic function. Genes encoding enzymes from all three classes have been identified in the prokaryotes with the beta and gamma classes predominating. All of the mammalian isozymes (including the 10 human isozymes) belong to the alpha class; however, only nine alpha class carbonic anhydrase genes have thus far been found in the Bacteria domain and none in the Archaea domain. The beta class is comprised of enzymes from the chloroplasts of both monocotyledonous and dicotyledonous plants as well as enzymes from phylogenetically diverse species from the Archaea and Bacteria domains. The only gamma class carbonic anhydrase that has thus far been isolated and characterized is from the methanoarchaeon Methanosarcina thermophila. Interestingly, many prokaryotes contain carbonic anhydrase genes from more than one class; some even contain genes from all three known classes. In addition, some prokaryotes contain multiple genes encoding carbonic anhydrases from the same class. The presence of multiple carbonic anhydrase genes within a species underscores the importance of this enzyme in prokaryotic physiology; however, the role(s) of this enzyme is still largely unknown. Even though most of the information known about the function(s) of carbonic anhydrase primarily relates to its role in cyanobacterial CO(2) fixation, the prokaryotic enzyme has also been shown to function in cyanate degradation and the survival of intracellular pathogens within their host. Investigations into prokaryotic carbonic anhydrase have already led to the identification of a new class (gamma) and future research will undoubtedly reveal novel functions for carbonic anhydrase in prokaryotes.
Carbonic anhydrases fall into three distinct evolutionary and structural classes: alpha, beta, and gamma. The beta-class carbonic anhydrases (beta-CAs) are widely distributed among higher plants, simple eukaryotes, eubacteria, and archaea. We have determined the crystal structure of ECCA, a beta-CA from Escherichia coli, to a resolution of 2.0 A. In agreement with the structure of the beta-CA from the chloroplast of the red alga Porphyridium purpureum, the active-site zinc in ECCA is tetrahedrally coordinated by the side chains of four conserved residues. These results confirm the observation of a unique pattern of zinc ligation in at least some beta-CAS: The absence of a water molecule in the inner coordination sphere is inconsistent with known mechanisms of CA activity. ECCA activity is highly pH-dependent in the physiological range, and its expression in yeast complements an oxygen-sensitive phenotype displayed by a beta-CA-deletion strain. The structural and biochemical characterizations of ECCA presented here and the comparisons with other beta-CA structures suggest that ECCA can adopt two distinct conformations displaying widely divergent catalytic rates.
The alpha-carbonic anhydrase gene from Helicobacter pylori strain 26695 has been cloned and sequenced. The full-length protein appears to be toxic to Escherichia coli, so we prepared a modified form of the gene lacking a part that presumably encodes a cleavable signal peptide. This truncated gene could be expressed in E. coli yielding an active enzyme comprising 229 amino acid residues. The amino acid sequence shows 36% identity with that of the enzyme from Neisseria gonorrhoeae and 28% with that of human carbonic anhydrase II. The H. pylori enzyme was purified by sulfonamide affinity chromatography and its circular dichroism spectrum and denaturation profile in guanidine hydrochloride have been measured. Kinetic parameters for CO2 hydration catalyzed by the H. pylori enzyme at pH 8.9 and 25 degrees C are kcat=2.4x10(5) s(-1), KM=17 mM and kcat/KM=1.4x10(7) M(-1) x s(-1). The pH dependence of kcat/KM fits with a simple titration curve with pK(a)=7.5. Thiocyanate yields an uncompetitive inhibition pattern at pH 9 indicating that the maximal rate of CO2 hydration is limited by proton transfer between a zinc-bound water molecule and the reaction medium in analogy to other forms of the enzyme. The 4-nitrophenyl acetate hydrolase activity of the H. pylori enzyme is quite low with an apparent catalytic second-order rate constant, k(enz), of 24 M(-1) x s(-1) at pH 8.8 and 25 degrees C. However, with 2-nitrophenyl acetate as substrate a k(enz) value of 665 M(-1) x s(-1) was obtained under similar conditions.
In the present paper, several points regarding Helicobacter pylori treatment are reviewed, with the following conclusions: (1) all different proton pump inhibitors (PPIs) are equivalent when prescribed with antibiotics; (2) ranitidine bismuth citrate is equal to or, in some cases with antibiotic resistance, more effective than PPI; (3) previous treatment with PPI does not seem to affect the rate of eradication obtained with PPI plus two antibiotics; (4) just 1 week of PPI is enough to obtain duodenal ulcer healing, provided that H. pylori eradication is achieved; (5) the eradication rates seem to be higher in peptic ulcer than in nonulcer dyspepsia; (6) in areas where the prevalence of metronidazole resistance is high, triple therapy including a PPI, clarithromycin, and amoxicillin is the best option, and (7) quadruple therapy (PPI, bismuth, tetracycline, and metronidazole) is the recommended second-line therapy after PPI-clarithromycin-amoxicillin failure, although replacing the PPI and the bismuth compound by ranitidine bismuth citrate achieves also good results.
Helicobacter pylori, the causative agent of peptic ulcer disease, expresses two different forms of the zinc-containing enzyme carbonic anhydrase (CA) (alpha and beta), catalyzing the reversible hydration of CO(2). Presumably, the high CO(2) requirement of H. pylori implies an important role for this enzyme in the bacterial physiology. In this paper, expression of the CAs has been analyzed in three different strains of the bacterium, 26695, J99 and 17.1, and appears to be independent of CO(2) concentration in the investigated range (0.1-10%). Presence of the potent and highly specific CA inhibitor, acetazolamide, in the medium does not seem to inhibit bacterial growth at the given sulfonamide concentration. Moreover, the localization and distribution of the alpha-CA was analyzed by immunonegative staining, while SDS-digested freeze-fracture immunogold labelling was used for the beta-form of the enzyme. The latter method has the advantage of allowing assessment of protein localization to distinct cell compartments and membrane structures. The resulting electron microscopy images indicate a localization of the beta-CA in the cytosol, on the cytosolic side of the inner membrane and on the outer membrane facing the periplasmic space. The alpha-enzyme was found attached to the surface of the bacterium.
To identify optimal antibiotics for second-line quadruple therapy of Helicobacter pylori after failed 1-week triple therapy.
One hundred patients were enrolled in this study after the failure of 1-week triple therapy. They were randomized to receive 1-week quadruple therapy consisting of amoxicillin, omeprazole and bismuth salts, plus either metronidazole or tetracycline. Before quadruple therapy, the H. pylori culture of each patient was tested for metronidazole resistance or clarithromycin resistance by E-test. Six weeks later, an endoscopy or 13C-urea breath test was used to define the success of H. pylori eradication.
The H. pylori eradication rates by intention-to-treat and per protocol analysis were higher in the tetracycline group than in the metronidazole group (intention-to-treat: 78% vs. 58%, P < 0.05; per protocol: 89% vs. 67%, P < 0.05). In the metronidazole group, but not in the tetracycline group, the per protocol eradication rate of quadruple therapy was lower for the infected isolates with metronidazole resistance than for those without metronidazole resistance (77% vs. 33%, P < 0.05).
Quadruple therapy, including tetracycline and amoxicillin, improves the H. pylori eradication rate after failed triple therapy.
To compare the efficacy of different regimens in patients in whom previous Helicobacter pylori eradication therapy has failed.
In this study named StratHegy patients (n=287) were randomized to receive one of three empirical triple therapy regimens or a strategy based on antibiotic susceptibility. The empirical regimens were omeprazole, 20 mg b.d., plus amoxicillin, 1000 mg b.d., and clarithromycin, 500 mg b.d., for 7 days (OAC7), clarithromycin, 500 mg b.d., for 14 days (OAC14) or metronidazole, 500 mg b.d., for 14 days (OAM14). In the susceptibility-based strategy, patients with clarithromycin-susceptible strains received OAC14, whilst the others received OAM14. The 13C-urea breath test was performed before randomization and 4-5 weeks after eradication therapy.
In the intention-to-treat analysis, the eradication rates for empirical therapies were as follows: OAC7, 47.4% (27/57); OAC14, 34.5% (20/58); OAM14, 63.2% (36/57); it was 74.3% (84/113) for the susceptibility-based treatment (P<0.01 when compared with OAC7 and OAC14). In patients receiving clarithromycin, the eradication rates were 80% for clarithromycin-susceptible strains and 16% for clarithromycin-resistant strains; in patients receiving OAM14, the eradication rates were 81% for metronidazole-susceptible strains and 59% for metronidazole-resistant strains.
Eradication rates of approximately 75% can be achieved with second-line triple therapy based on antibiotic susceptibility testing. If susceptibility testing is not available, OAM14 is an appropriate alternative.
Owing to rising drug-resistant Helicobacter pylori infections, currently recommended proton-pump inhibitor-based triple therapies are losing their efficacy, and regimens efficacious in the presence of drug resistance are needed.
To summarize the efficacy, safety and adherence of first-line quadruple H. pylori therapies in adults.
Meta-regression models identified factors explaining variation in the efficacy of first-line quadruple therapies from 145 treatment arms. Estimates of average efficacy were calculated within homogeneous groups.
Quadruple therapy containing a gastric acid inhibitor, bismuth, metronidazole and tetracycline was enhanced when omeprazole was included, treatment duration lasted 10-14 days, and when therapy took place in the Netherlands, Hong Kong and Australia. Treatment efficacy decreased as the prevalence of metronidazole resistance increased. Even in areas with a high prevalence of metronidazole resistance, this quadruple regimen eradicated more than 85% of H. pylori infections when it contained omeprazole and was given for 10-14 days. Furthermore, in the presence of clarithromycin resistance, this quadruple regimen eradicated 90-100% of H. pylori infections, while the currently recommended triple therapy containing clarithromycin, amoxicillin and a proton-pump inhibitor eradicated only 25-61% (P < 0.001). Adherence and adverse events for quadruple therapy were similar to currently recommended triple therapies.
Guidelines should include quadruple therapy with a proton-pump inhibitor, a bismuth compound, metronidazole and tetracycline among recommended first-line anti-H. pylori therapies.
Carbonic anhydrase inhibitors have been successfully used to treat peptic ulcers. Although carbonic anhydrase restriction does not inhibit Helicobacter pylori in vitro, recent studies suggest that carbonic anhydrase inhibition reduces the ability of H. pylori to survive in an acid environment as present in the stomach.
In a pilot study, we examined the effect of acetazolamide 500 mg as twice a day for 4 days in volunteers with active H. pylori infection. Effectiveness was judged by changes in the results of the urea breath test.
Eight H. pylori infected volunteers completed the test. No urea breath test reverted to negative and there was a trend for the urea breath test value to increase [e.g. delta over baseline (DOB) mean +/- SE increased from 50.9 +/- 13 at baseline to 64.9 +/- 13 at day 5] during treatment with acetazolamide.
The potential effect of carbonic anhydrase inhibitors on acid secretion may prevent effect on H. pylori in vivo and/or the sites of infection at the surface of the stomach may have a pH higher for any postulated acid-dependent effect to have an effect clinically.
In the gastric microenvironment, Helicobacter pylori is exposed to bicarbonate, urea and acid. Here it is demonstrated that both H. pylori carbonic anhydrases (CAs) are required for maintaining urease activity and therefore influence H. pylori urea resistance at neutral pH. Furthermore, the beta-CA is required for acid resistance as indicated by a growth defect of the corresponding mutant at low pH. The alpha- and beta-CA mutants as well as the double mutant were more resistant to bicarbonate, indicating that both enzymes are involved in bicarbonate metabolism. These phenotypes support important CA-functions in H. pylori urea and bicarbonate metabolism and acid resistance. Thus, both CA enzymes might be required for survival in the gastric niche.
To evaluate the efficacy of omeprazole triple therapy versus omeprazole quadruple therapy for Helicobacter pylori infection.
Prospective, randomized, single-centre, investigator-blind study.
Departments of Gastroenterology and Histopathology, Evangelismos Hospital, Athens, Greece.
One hundred and forty-nine consecutive patients with active duodenal ulcer were randomized to receive omeprazole (20 mg b.d.), amoxicillin (1 g b.d.) and clarithromycin (0.5 g b.d.) (OAC, n = 78), or omeprazole (20 mg b.d.), colloidal bismuth subcitrate (120 mg q.i.d.), metronidazole (0.5 g t.i.d.) and tetracycline hydrochloride (0.5 g q.i.d.) (OBMT, n = 71) for 10 days. Patients' symptoms were scored, and compliance and treatment-related side effects were assessed. Endoscopy was performed before treatment and at 10-12 weeks and 12 months after treatment. H. pylori infection and its successful eradication were sought by histology, immunohistochemistry and campylobacter-like organisms (CLO) tests on multiple biopsies taken from the gastric antrum, corpus and fundus. Patients were re-evaluated clinically and underwent a C-urea breath test (UBT) at 21-24 months. Those with dyspepsia and/or recrudescence of H. pylori were re-endoscoped.
Patient groups were comparable for age, sex, smoking, occasional use of nonsteroidal anti-inflammatory drugs (NSAIDs), and current or past bleeding episodes. Six and seven patients in the OAC and OBMT treatment groups, respectively, were lost to follow-up. Eight patients were non-compliant. Two ulcers in the OAC group and one in the OBMT group did not heal. By intention-to-treat (ITT) and per-protocol (PP) analyses, ulcer healing rates were 86% (67/78) and 97% (67/69), respectively, for the OAC group, and 82% (58/71) and 98% (58/59), respectively, for the OBMT group. H. pylori eradication at 10-12 weeks after treatment was 78% (61/78) and 88% (61/69) for OAC, and 65% (46/71) and 78% (46/59) for OBMT, by ITT and PP analyses, respectively (P > 0.1). Side effects were more common with OBMT. Relapse rates of H. pylori were 3% and 2% for the first and second years, respectively. Four H. pylori-negative patients developed reflux symptoms, but only two developed erosive oesophagitis between 12 and 24 months.
OAC and OBMT were equally effective in healing active duodenal ulcers and eradicating H. pylori, but OAC should be used as a first-line treatment because of its better tolerance.
The efficacy of established Helicobacter pylori regimes needs to be reviewed. In view of drug resistance, side effects, and compliance and expense of therapy, treatment failure is increasing and second-line treatment strategies need to be developed. A simulation model suggested by the Cochrane review group showed that H. pylori eradication is cost-effective for duodenal and gastric ulcer long-term. The duration of eradication therapy continues to be controversial. In Europe and other parts of the world, 7-day triple regimes are used, whereas guidelines from the United States recommend 10-14 days of therapy. Antibiotic resistance is a major factor affecting the outcome of eradication therapy. New modified eradication regimes involve substitution of antibiotics used in conjunction with other drugs. The newer generation fluoroquinolones have shown some promise as part of an eradication regimen. Quadruple therapy (bismuth, proton pump inhibitor [PPI] and two antibiotics and sequential treatment [PPI with three antibiotics]) are promising first-line treatments. Novel agents have been tried, but with disappointing results. New drugs and administration forms have been reported but their efficacy needs confirmation.
Helicobacter pylori is a Gram-negative neutralophile associated with peptic ulcers and gastric cancer. It has a unique ability to colonize the human stomach by acid acclimation. It uses the pH-gated urea channel, UreI, to enhance urea access to intrabacterial urease and a membrane-anchored periplasmic carbonic anhydrase to regulate periplasmic pH to approximately 6.1 in acidic media, whereas other neutralophiles cannot regulate periplasmic pH and thus only transit the stomach.
A library of sulfonamides/sulfamates has been investigated for the inhibition of the carboxyterminal truncated form of the alpha-carbonic anhydrase (CA, EC 4.2.1.1) isolated from the gastric pathogen Helicobacter pylori (hpCA). This enzyme, incorporating 202 amino acid residues, showed a catalytic activity similar to that of the full length hpCA, with k(cat) of 2.35 x 10(5)s(-1) and k(cat)/K(M) of 1.56 x 10(7)M(-1)s(-1) at 25 degrees C and pH of 8.9, for the CO(2) hydration reaction. All types of activity for inhibition of the bacterial enzyme have been detected. Dorzolamide and simple 4-substituted benzenesulfonamides were weak hpCA inhibitors (inhibition constants, K(I)s, in the range of 830-4310 nM). Sulfanilamide, orthanilamide, some of their derivatives, and indisulam showed better activity (K(I)s in the range of 310-562 nM), whereas most of the clinically used CA inhibitors, such as methazolamide, ethoxzolamide, dichlorophenamide, brinzolamide, topiramate, zonisamide, etc., acted as medium potency hpCA inhibitors (K(I)s in the range of 124-287 nM). Some potent hpCA inhibitors were detected too (K(I)s in the range of 20-96 nM) such as acetazolamide, 4-amino-6-chloro-1,3-benzenedisulfonamide, 4-sulfanilyl-aminoethyl-benzenesulfonamide, and 4-(2-amino-pyrimidin-4-yl)-benzenesulfonamide. Most of the investigated derivatives acted as better inhibitors of the human isoform hCA II than as hpCA inhibitors. Since hpCA is essential for the survival of the pathogen in acid, its inhibition by compounds such as those investigated here might be used as a new pharmacologic tool in the management of drug resistant H. pylori.
We have cloned and sequenced Helicobacter pylori alpha-class carbonic anhydrase (hpCA) from patients with different gastric mucosal lesions, including gastritis (n=15), ulcer (n=6), and cancer (n=16). Although several polymorphisms were newly identified such as 12Ala, 13Thr, 16Ile, and 168Phe, there was no significant relevance of any polymorphism with gastric mucosal lesion types. A library of sulfonamides/sulfamates has been investigated for the inhibition of hpCA, whereas new derivatives have been obtained by attaching 4-tert-butyl-phenylcarboxamido/sulfonamido tails to benzenesulfonamide/1,3,4-thiadiazole-2-sulfonamide scaffolds. All types of activity for inhibition of hpCA have been detected. Dorzolamide and simple 4-substituted benzenesulfonamides were weak inhibitors (KI 873-4360 nM). Sulfanilamide, orthanilamide, some of their derivatives, and indisulam showed better activity (KI 413-640 nM), whereas most of the clinically used inhibitors, such as methazolamide, ethoxzolamide, dichlorophenamide, brinzolamide, topiramate, zonisamide, etc., acted as medium-potency inhibitors (KI 105-378 nM). Some potent hpCA inhibitors were detected too (KI 12-84 nM) among acetazolamide, 4-amino-6-chloro-1,3-benzenedisulfonamide and some newly designed compounds incorporating lipophilic tails. Some of the newly prepared derivatives had selectivity ratios for inhibiting hpCA over hCA II in the range of 1.25-3.48, showing thus some selectivity for inhibiting the bacterial enzyme. Since hpCA is essential for the survival of the pathogen in acid, it might be used as a new pharmacologic tool in the management of drug-resistant H. pylori.
Activation of six human carbonic anhydrases (CA, EC 4.2.1.1), that is, hCA I, II, IV, VA, VII, and XIV, with l- and d-histidine was investigated through kinetics and by X-ray crystallography. l-His was a potent activator of isozymes I, VA, VII, and XIV, and a weaker activator of hCA II and IV. d-His showed good hCA I, VA, and VII activation properties, being a moderate activator of hCA XIV and a weak activator of hCA II and IV. The structures as determined by X-ray crystallography of the hCA II-l-His/d-His adducts showed the activators to be anchored at the entrance of the active site, contributing to extended networks of hydrogen bonds with amino acid residues/water molecules present in the cavity, explaining their different potency and interaction patterns with various isozymes. The residues involved in l-His recognition were His64, Asn67, Gln92, whereas three water molecules connected the activator to the zinc-bound hydroxide. Only the imidazole moiety of l-His interacted with these amino acids. For the d-His adduct, the residues involved in recognition of the activator were Trp5, His64, and Pro201, whereas two water molecules connected the zinc-bound water to the activator. Only the COOH and NH(2) moieties of d-His participated in hydrogen bonds with these residues. This is the first study showing different binding modes of stereoisomeric activators within the hCA II active site, with consequences for overall proton-transfer processes (rate-determining for the catalytic cycle). The study also points out differences of activation efficiency between various isozymes with structurally related activators, convenient for designing alternative proton-transfer pathways, useful both for a better understanding of the catalytic mechanism and for obtaining pharmacologically useful derivatives, for example, for the management of Alzheimer's disease.
Carbonic anhydrases (CAs, EC 4.2.1.1) are zinc enzymes acting as efficient catalysts for the reversible hydration of carbon dioxide to bicarbonate. 16 different alpha-CA isoforms were isolated in mammals, where they play crucial physiological roles. Some of them are cytosolic (CA I, CA II, CA III, CA VII, CA XIII), others are membrane-bound (CA IV, CA IX, CA XII, CA XIV and CA XV), CA VA and CA VB are mitochondrial, and CA VI is secreted in saliva and milk. Three acatalytic forms are also known, the CA related proteins (CARP), CARP VIII, CARP X and CARP XI. Representatives of the beta-delta-CA family are highly abundant in plants, diatoms, eubacteria and archaea. The catalytic mechanism of the alpha-CAs is understood in detail: the active site consists of a Zn(II) ion co-ordinated by three histidine residues and a water molecule/hydroxide ion. The latter is the active species, acting as a potent nucleophile. For beta- and gamma-CAs, the zinc hydroxide mechanism is valid too, although at least some beta-class enzymes do not have water directly coordinated to the metal ion. CAs are inhibited primarily by two classes of compounds: the metal complexing anions and the sulfonamides/sulfamates/sulfamides possessing the general formula RXSO(2)NH(2) (R=aryl; hetaryl; perhaloalkyl; X=nothing, O or NH). Several important physiological and physio-pathological functions are played by CAs present in organisms all over the phylogenetic tree, related to respiration and transport of CO(2)/bicarbonate between metabolizing tissues and the lungs, pH and CO(2) homeostasis, electrolyte secretion in a variety of tissues/organs, biosynthetic reactions, such as the gluconeogenesis and ureagenesis among others (in animals), CO(2) fixation (in plants and algae), etc. The presence of these ubiquitous enzymes in so many tissues and in so different isoforms represents an attractive goal for the design of inhibitors with biomedical applications. Indeed, CA inhibitors are clinically used as antiglaucoma drugs, some other compounds being developed as antitumour agents/diagnostic tools for tumours, antiobesity agents, anticonvulsants and antimicrobials/antifungals (inhibitors targeting alpha- or beta-CAs from pathogenic organisms such as Helicobacter pylori, Mycobacterium tuberculosis, Plasmodium falciparum, Candida albicans, etc.).
DNA clones for the beta-class carbonic anhydrase (CA, EC 4.2.1.1) of Helicobactor pylori (hpbetaCA) were obtained. A recombinant hpbetaCA protein lacking the N-terminal 15-amino acid residues was produced and purified, representing a catalytically efficient CA. hpbetaCA was strongly inhibited (K(I)s in the range of 24-45 nM) by many sulfonamides/sulfamates, among which acetazolamide, ethoxzolamide, topiramate, and sulpiride, all clinically used drugs. The dual inhibition of alpha- and/or beta-class CAs of H. pylori might represent a useful alternative for the management of gastritis/gastric ulcers, as well as gastric cancer. This is also the first study showing that a bacterial beta-CA can be a drug target.
Three benzene-1,3-disulfonamide derivatives were investigated for their interaction with 12 mammalian alpha-carbonic anhydrases (CAs, EC 4.2.1.1), and three bacterial/archaeal CAs belonging to the alpha-, beta-, and gamma-CA class, respectively. X-ray crystal structure of the three inhibitors in complex with the dominant human isozyme CA II revealed a particular binding mode within the cavity. The sulfonamide group in meta-position to the Zn(2+)-coordinated SO(2)NH(2) moiety was oriented toward the hydrophilic side of the active site cleft, establishing hydrogen bonds with His64, Asn67, Gln92, and Thr200. The plane of the phenyl moiety of the inhibitors was rotated by 45 degrees and tilted by 10 degrees with respect to its most recurrent orientation in other CA II-sulfonamide complexes.